title: Phosphorylation-independent activity of the response regulators AlgB and AlgR in promoting alginate biosynthesis in mucoid Pseudomonas aeruginosa.
abstract: Overproduction of the capsular polysaccharide alginate appears to confer a selective advantage for Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The regulators AlgB and AlgR, which are both required as positive activators in alginate overproduction, have homology with the regulator class of two-component environmental responsive proteins which coordinate gene expression through signal transduction mechanisms. Signal transduction in this class of proteins generally occurs via autophosphorylation of the sensor kinase protein and phosphotransfer from the sensor to a conserved aspartate residue, which is present in the amino terminus of the response regulator. Recently, kinB was identified downstream of algB and was shown to encode the cognate histidine protein kinase that efficiently phosphorylates AlgB. However, we show here that a null mutation in kinB in a mucoid cystic fibrosis isolate, P. aeruginosa FRD1, did not block alginate production. The role of the conserved aspartate residue in the phosphorylation of AlgB was examined. The predicted phosphorylation site of AlgB (D59) was mutated to asparagine (N), and a derivative of an AlgB lacking the entire amino-terminal phosphorylation domain (AlgB delta1-145) was constructed. A hexahistidine tag was included at the amino terminus of the wild-type (H-AlgB), H-AlgB delta1-145, and mutant (H-AlgB.59N) AlgB proteins. These derivatives were purified by Ni2+ affinity chromatography and examined for in vitro phosphorylation by the purified sensor kinase protein, KinB. The results indicated that while KinB efficiently phosphorylated H-AlgB, no phosphorylation of H-AlgB delta1-145 or H-AlgB.D59N was apparent. An allelic exchange system was developed to transfer mutant algB alleles onto the chromosome of a P. aeruginosa algB mutant to examine the effect on alginate production. Despite the defect in AlgB phosphorylation, P. aeruginosa strains expressing AlgB.D59N or H-AlgB delta1-145 remained mucoid. The roles of the conserved aspartate residues in the phosphorylation of AlgR were also examined. As seen with AlgB, mutations in the predicted phosphorylation site of AlgR (AlgR.D54N and AlgR.D85N) did not affect alginate production. These results indicate that in vivo phosphorylation of AlgB and AlgR are not required for their roles in alginate production. Thus, the mechanism by which these response regulators activate alginate genes in mucoid P. aeruginosa appears not to be mediated by conventional phosphorylation-dependent signal transduction.
authors.affiliation: Department of Microbiology and Immunology, University of Tennessee and Veterans Administration Medical Center, Memphis 38163, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ma
authors.forename: U
authors.initials: U
authors.lastname: Selvaraj
authors.forename: D E
authors.initials: DE
authors.lastname: Ohman
authors.forename: R
authors.initials: R
authors.lastname: Quarless
authors.forename: D J
authors.initials: DJ
authors.lastname: Hassett
authors.forename: D J
authors.initials: DJ
authors.lastname: Wozniak
mesh_terms: D000464:Alginates / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D009876:Operon; D010766:Phosphorylation; D010770:Phosphotransferases; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D015398:Signal Transduction; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: C110681:AlgR protein, Pseudomonas aeruginosa; D000464:Alginates; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011135:Polysaccharides, Bacterial; D015534:Trans-Activators; D014157:Transcription Factors; C072862:AlgB protein, Pseudomonas; D010770:Phosphotransferases; D011494:Protein Kinases; C081245:kinB protein, Bacillus subtilis